Effects of different inspiratory muscle warm-up loads on mechanical, physiological and muscle oxygenation responses during high-intensity running and recovery

Inspiratory muscle warm-up (IMW) has been used as a resource to enhance exercises and sports performance. However, there is a lack of studies in the literature addressing the effects of different IMW loads (especially in combination with a shorter and applicable protocol) on high-intensity running and recovery phase. Thus, this study aimed to investigate the effects of three different IMW loads using a shorter protocol on mechanical, physiological and muscle oxygenation responses during and after high-intensity running exercise. Sixteen physically active men, randomly performed four trials 30 s all-out run, preceded by the shorter IMW protocol (2 × 15 breaths with a 1-min rest interval between sets, accomplished 2 min before the 30 s all-out run). Here, three IMW load conditions were used: 15%, 40%, and 60% of maximal inspiratory pressure (MIP), plus a control session (CON) without the IMW. The force, velocity and running power were measured (1000 Hz). Two near-infrared spectroscopy (NIRS) devices measured (10 Hz) the muscle’s oxygenation responses in biceps brachii (BB) and vastus lateralis (VL). Additionally, heart rate (HR) and blood lactate ([Lac]) were also monitored. IMW loads applied with a shorter protocol promoted a significant increase in mean and minimum running power as well as in peak and minimum force compared to CON. In addition, specific IMW loads led to higher values of peak power, mean velocity (60% of MIP) and mean force (40 and 60% of MIP) in relation to CON. Physiological responses (HR and muscles oxygenation) were not modified by any IMW during exercise, as well as HR and [Lac] in the recovery phase. On the other hand, 40% of MIP presented a higher tissue saturation index (TSI) for BB during recovery phase. In conclusion, the use of different loads of IMW may improve the performance of a physically active individual in a 30 s all-out run, as verified by the increased peak, mean and minimum mechanical values, but not in performance assessed second by second. In addition, 40% of the MIP improves TSI of the BB during the recovery phase, which can indicate greater availability of O2 for lactate clearance.

Participants. Sixteen physically active young men (local sports team players and street running participants) were evaluated (23 ± 1 years old, 73.2 ± 2.0 kg; 177.4 ± 1.9 cm; 9.0 ± 0.5% body fat, IPAQ at 4539 ± 942 metabolic equivalent-min/week; MIP at 145.6 ± 9.9 cmH 2 O, peak of global strength index (S-Index) at 139 ± 3 and mean S-Index at 123 ± 4, cmH 2 O). The analysis with G*Power software showed that a sample size of at least 12 individuals would be necessary to obtain a power of 80% with a significance level α = 5%, based on previously published data 10 . The participants were invited to answer questionnaires about their levels of physical activity (International Physical Activity Questionnaire-IPAQ), sports practice and health history. Only participants that presented a minimum score to classify them as 'physically active' were included in the study 42 . Individuals that reported metabolic, cardiovascular, respiratory or orthopedic disease were excluded from this research.  (Fig. 1). On the first day, after signing the informed consent form and completing the questionnaires, anthropometric and body composition measurements were performed. In the second visit, MIP and S-Index were determined 1 h apart to prevent inspiratory fatigue. On the same day, the participants were familiarized with the inspiratory muscle warm-up protocol and the non-motorized treadmill (NMT), when they were asked to perform five sprints of 10 s. In the next four visits to the laboratory, all participants were submitted to high-intensity tethered exercise (30 s all-out run), preceded by different IMW loads protocol (15%, 40% and 60% of MIP plus a control (CON) session without the IMW protocol). These sessions were randomly performed by the individuals under the IMW protocols used. Upon arrival at the laboratory, the participants were equipped with near-infrared spectroscopy (NIRS) devices attached to the biceps brachii (BB) and vastus lateralis (VL) muscles, and a heart rate monitor (HR) for data acquisition throughout the session. The participants remained at rest for 3 min for the determination of baseline values (BL), including blood lactate concentration ([Lac]) at rest. Then, they were asked to warm-up on a motorized treadmill for 5 min (7 km/h and 1% inclination) and rest for another 5 min. Subsequently, the IMW protocol was performed. Two minutes after, the individuals were submitted to 30 s high-intensity tethered running for data acquisition (i.e., force, velocity and running power). Immediately after the test (T0), blood samples were collected every 2 min up to 18 min of passive recovery (T2-T18). In all sessions, the participants were instructed to have a light meal, not to consume alcohol/caffeine and not to practice moderate-intense exercise 24 h before the tests. The procedures were performed in a controlled and isolated laboratory environment, and the participants did not receive any information about each intervention.  www.nature.com/scientificreports/ Inspiratory measurements and inspiratory muscle warm-up. The analysis was conducted by a trained researcher who demonstrated the correct performance of the respiratory maneuver. The participants remained seated on a chair, wearing a nose clip and a plastic mouthpiece connected to an analogical manovacuometer (± 300 cmH 2 O; GER-AR, São Paulo, SP, Brazil) used to measure maximal pressures. A small hole (2 mm) was introduced in the rigid mouthpiece in order to prevent glottic closure. The participants were instructed to complete three to five acceptable and reproducible maximum maneuvers (i.e., differences of 10% or less between values), with 1 min interval between maneuvers 43,44 . Each inspiratory effort was sustained for at least 1 s, and the MIP was considered the highest value between these attempts 45 . After 1 h, a dynamic proposal to characterize the strength of the participants' IM, the global strength index (S-Index), was assessed by an inspiratory threshold (POWERbreathe K5, IMT Technologies Ltd., Birmingham, UK), with the participants in standing position and using a nose clip. Thirty dynamic inspirations resistancefree were performed slowly, with verbal encouragement to inhale a greater air capacity 32 . During the protocol, breathing pattern curves were monitored by graphic records provided by Breathe-link® software. At the end of the test, the algorithm provided the mean and peak values of the S-Index (in units of cmH 2 O).
The inspiratory muscle warm-up (IMW) protocol loads were also applied using the inspiratory device POW-ERbreathe K5. The participants initiated every breath from residual volume and were encouraged to continue the respiratory effort until further excursion of the thorax was not possible, with a diaphragmatic breathing pattern. Subsequently, they were instructed to keep the same inspiratory pressure and the breathing pattern curves were also monitored by Breathe-link® software. The total protocol was comprised of two sets of 15 inspirations with a 1-min rest interval between them. The loads were equivalent to 15% (WU15), considered placebo by the literature 32,35,39 , 40% (WU40) and 60% (WU60) of MIP. All experimental trials were randomly distributed.
The 30 s all-out run test and mechanical measurements. The 30 s all-out run was performed on a non-motorized treadmill (NMT) (Inbramed Super ATL, Inbrasport, Porto Alegre, Brazil), as detailed by Manchado-Gobatto et al. 10 . Two minutes after the IMW or CON protocols, the participants were asked to run at maximum intensity for 30 s, tethered by their waist to an inextensible steel cable attached to a load cell (CSL/ ZL-250, MK Controle e Instrumentação Ltda, Brazil) for horizontal force measurement. Other four load cells (CSAL/ZL-500, MK Controle e Instrumentação Ltda, Brazil) were positioned under the NMT platform to measure the vertical force (signal frequency at 1000 Hz). A hall-effect sensor in the frontal axis of the NMT provided pulses for velocity acquisition. Therefore, both vertical and horizontal force components were measured during the running exercise along with velocity to calculate the power running. The signals were synchronized and the product between force and velocity resulted in the running power, with the peak, mean and minimum values relativized to body mass. Fatigue index (FI) was also calculated by the following equation: FI = (peak powerminimum power)/peak power * 100). The system was calibrated on the test days.

Physiological responses. Blood lactate concentration and heart rate. For lactate concentrations ([Lac])
at rest, post-effort and every 2 min up to 18 min of passive recovery, blood samples (25 µl) were collected from the participants' earlobe with heparinized capillaries, deposited in microtubes (Eppendorf, 1.5 ml containing 50 µl of 1% sodium fluoride-NaF) and frozen at − 20 °C. The [Lac] were determined by a lactate analyzer (YSI-2300-STAT-Plus™, Yellow Springs, USA). Throughout the protocols, the heart rate (HR) was constantly recorded (at 1 Hz) (Polar V800, Finland). For all variables, the peak, mean and minimum values were calculated (during the test we used the 30 s responses, while during passive recovery we considered the mean of 18 min).  46 throughout the experimental protocol by two PortaMon devices (Artinis, Medical Systems BV, Zetten, Netherlands) working on the modified Beer-Lambert law. Each device has three light source transmitters (with two wavelengths of 760 and 850 nm), positioned at 30, 35 and 40 mm from the receiver. The devices were safely fixed and covered to eliminate background light after shaving and cleaning the skin surface. While one was positioned in the medial part (belly) of the BB 10,37,47 of the right arm, considered less active during running, the other was allocated in the VL of the right leg (considered more active), 15 cm above the proximal edge of the patella and 5 cm to the external side 10,48-50 . Skinfolds for BB (3.3 ± 0.2 mm) and VL (11.2 ± 1.2 mm) were less than half the distance between the source and the deepest detector (i.e., 20 mm). Different path lengths (DPF) were used for BB (3.78) and VL (3.83) 10 . The signals were smoothed using a 10 th order low-pass zero-phase Butterworth filter (cutoff frequency of 0.1 Hz) 50  There was an increase in running power in the first second until a peak power was reached at approximately 6 s and a consequent decrease after this time for all interventions, without any effects of IMW on the studied parameter. Panels B, C, D and E display the peak, mean and minimum values for power, force, velocity and FI, respectively. For these measurements, the one-way repeated measures ANOVA revealed an effect of IMW (F (3,45)

Discussion
To the best of our knowledge, this is the first study dedicated to investigating the effects of different IMW loads (15,40 and 60% of MIP) on mechanical and physiological responses, including oxygenation in more and less active muscles, during and after high-intensity, short-duration running exercise. Additionally, we studied these acute inspiratory strategies using a shorter protocol (i.e., lower number of exercise repetitions). Our main findings revealed some effects of IMW, performed with 2 sets of 15 repetitions with a 1-min rest interval between the sets, on the high-intensity running effort and recovery, independently of the load applied. Regarding the mechanical parameters, all IMW promoted a significant increase in mean and minimum running power, as well as in peak and minimum running force compared to CON. Additionally, when applying specific IMW loads higher values were observed for peak power, mean velocity (WU60) and mean force (WU40 and WU60) in relation to CON. The physiological responses, including HR and oxygenation in more and less active muscles during the running exercise, were not modified by IMW, at least not during the 30 s high-intensity running nor for HR and [Lac] in the post-effort phase. By comparing the responses in BB and VL, no differences were observed in mechanical and muscle oxygenation during the 30 s all-out test. In passive recovery, higher TSI values for VL were detected in the post-effort phase (T0) for all protocols. An interesting finding was observed in WU40, which reached www.nature.com/scientificreports/ higher oxygenation values from T4 to T10 compared to BL. It can be then suggested that the use of different loads of IMW promotes an improvement in performance corroborated by the increased peak, mean and minimum mechanical values, but not in the performance assessed second by second. Also, WU40 may improve recovery phase with higher oxygenation in BB.

Inspiratory muscle warm-up and performance. Our choice to investigate the effect of different IMW
loads on the performance of high-intensity, short-duration running exercise was based on previous studies that indicate the positive effect of IMW, but used different IWM protocols in intermittent running 27 , in Wingate tests 33,34 , in 100 m freestyle swimming 29 , in specific hockey drills 31 and in a simulate judo match 32 . Additionally, considering the large use of tethered efforts in physical and sports programs together with the significance of high-intensity exercise in this context, we focused on the evaluation of the IMW impact on the force, velocity and running power of 30 s all-out run sessions using different inspiratory loads. As shown in Fig. 2, the same characteristics were observed for the curve of running power throughout the tests, with no differences among the IMW load interventions during the 30 s all-out run sessions. As previously mentioned, running power, force and velocity were improved by the IMW loads, more specifically the WU40 and WU60, which significantly influenced the mechanical variables compared to CON, suggesting an improvement in running performance for active participants. Regarding the exercise performance of athletes, the IMW combined with specific warm-up was capable of reducing the time in 100 m freestyle swimming 29 , treadmill sprint performance 51 and interactions among the technical-tactical, physical, physiological, and psychophysiological parameters in a simulated judo match 32 . Studies that used IMW as the only means of prior effort to main motor task also observed a reduction in the sensation of dyspnea and an improvement in the distance walked in one badminton-footwork test 36 , as well as in one shuttle run test 52 . Similarly, Özdal and colleagues 34 observed an increase in peak and relative power in   28,53 , on performance in a knee flexion-extension protocol accomplished in an isokinetic test by healthy sedentary participants 41 and on long-distance test 30 were previously observed. On the other hand, other studies did not find significant effects of IMW for both active individuals and athletes performing different types of exercise and tests 39,43,46,[53][54][55][56] . It is important to consider the diversity of the IMW protocols when applied to different populations, sports modalities and exercise tests, which makes it difficult to compare the results obtained with other findings. Moreover, most investigations do not describe the time interval between the IMW sets and the time between the IMW application and the test or main exercise. Knowing that the effects of warm-up can be affected by several factors, such as protocol, load, performance level, type of exercise, time interval between the conditioning stimulus and the performance testing, etc. 57,58 , more attention could be paid to these aspects. In this sense, we focused on a shorter IMW protocol (2 sets of 15 repetitions with a 1-min rest interval between them, concluded 2 min before the running test), using different inspiratory loads in each session (without and with 15, 40 and 60% of MIP) applied to active participants. Regarding the respiratory parameters, even though our participants did not have any experience with respiratory training or inspiratory warm-up, they reached good MIP values (145.6 ± 9.9 cmH 2 O), similar to Japanese athletes in triathlon and wrestling (light category) modalities (145.8 and 147.3 cmH 2 O, respectively) 59 .
Inspiratory muscle warm-up and physiological responses. The ventilatory responses in high-intensity exercises can affect the perfusion dynamics of the locomotor muscles and tissue saturation indices, representing a limitation of exercise performance 19,20,60 . According to the literature, inspiratory muscle warm-up can be a strategy to potentialize oxygenation redistribution to more active muscles during physical exercise 35 . However, improvements in post-effort recovery process remain unexplored. Thus, we measured for the first time the physiological responses in exercise and recovery, including oxygenation analysis in more or less active muscles (which are relevant to providing oxygen and removing metabolites, respectively). Few scientific investigations, especially in sports, have been conducted to study the IMW potential to minimize respiratory fatigue and improve the oxygenation redistribution in high-intensity exercise 35,40,46,51,61 . When studying the oxygenation in the gastrocnemius muscle of female soccer players by submitting them to submaximal cycling test and intermittent cycling test, Cheng et al. 35 demonstrated that the IMW protocol can enhance oxygen saturation in this tissue. However, the authors did not observe changes in performance, possibly due to the lack of specificity in the test for these athletes.
In our study, second-by-second NIRS analyses did not reveal the effects of IMW on muscle responses, regardless of the load (Fig. 3, Panels A-B). Despite the evidence of limb muscle effect on TSI peak, mean and minimum values (Fig. 3, Panel C) and [tHb] mean and minimum values (Fig. 3, Panel F), no interaction with IMW protocols was observed, indicating a similar behavior in BB and VL oxygenation. Whether the IMW promotes a positive effect on the oxygenation redistribution 35 , this was not observed in more and less active muscles. Considering the increased respiratory muscle work and the competition with locomotor muscles for O 2 supply 17-20 , the analysis of inspiratory muscle oxygenation could provide some insight into the oxygenation between these muscles. However, the analysis of oxygenation occurs only in accessory and secondary inspiratory muscles 62 , and it does not directly reflect the oxygenation of the muscle with the greatest potential for oxygen uptake and the most affected one by IMW, the diaphragm. Recently, studies addressing the IMW applied to speed skaters on ice time trial also did not report any improvement in muscle oxygenation variables in the right VL, with some limitations pointed out by the authors, such as leg compression garments and small sample size 40,46 . On the other hand, in high-intensity sprint 10 and high-intensity cycling 37 a difference in more and less active muscles was observed, suggesting adjustments in oxygenation during effort in a tissue-dependent manner.
In order to support the high demand of the respiratory muscles during exercise, the VO 2 and oxygenation in this region are increased, and may compromise cardiac output by 14-16% in well-trained individuals 63 , thus affecting oxygenation distribution to locomotor muscles 64 . In high-intensity effort, these locomotor muscles also use predominantly anaerobic pathways, resulting in lactate production. According to previous studies, inspiratory muscles may play an important role as lactate consumers 21,22 . In this context, Lin et al. 36 indicated a reduction in [Lac] in badminton players after IMW. Regarding [Lac], the peak values observed here (~ 16 mM) confirmed their significant anaerobic contribution in the 30 s all-out run. These findings corroborate previous studies on exercises characterized by anaerobic contribution 9,10,65,66 . In our study, both HR and [Lac] were not affected by IMW interventions, and 18 min post-effort in passive recovery was not sufficient to make these physiological responses return to baseline values (Fig. 4, Panels A-D).
With respect to passive recovery, a higher decrement in oxygenation was observed immediately after the exercise, the so-called post-effort phase (T0), with quick adjustments after T2 for both muscles (Fig. 5). Osawa et al. 37 reported that tissue oxygenation did not begin immediately after high-intensity cycling effort and that deoxygenation occurred for a few seconds. In the present study, TSI percentages started to rise immediately after the exercise, and after 4 min (T4) they returned to baseline values (Fig. 5, Panels A-B). Interestingly, only WU40 presented higher TSI values in BB from T4 to T10 in relation to BL. We did not perform correlation analyses, however, Manchado-Gobatto et al. 10  www.nature.com/scientificreports/ addition to a better blood lactate removal during recovery in relation to other loads, we observed that different inspiratory loads can improve mechanical parameters and recovery oxygenation. Recent studies investigated the effects of IMW as a warm-up strategy combined with core warm-ups on recovery period between intermittent exercise and repeated sprints on NMT 51 and on recovery periods of sprints on a cycle ergometer 68 . Although the authors evaluated recovery, muscle oxygenation was only observed during exercise. In our study, the comparison between BB and VL revealed that only immediately after the 30 s all-out run the VL presented higher values for all interventions (Fig. 5, Panels A-B). These results corroborate those reported by Manchado-Gobatto et al. 10 , who did not use inspiratory strategy to improve the running performance. Finally, the responses during exercise and recovery are a complex process. Thus, to improve the interpretation of IMW on running and recovery, integrative analyses could reveal responses beyond conventional statistics. For example, our group recently observed the improvement in the technical and tactical parameters in a judo simulated fight using the same shorter IMW protocol used herein, based on a complex network analysis 32 . In such study, the centrality metrics revealed that the IWM at 15% of MIP favored the interactions among the psychophysiological, physical and physiological parameters, while the IWM at 40% of MIP was able to improve performance in the judo match. Therefore, our next investigations will be considering these findings.
Furthermore, a recent study indicated NIRS measurements as a future physiological marker, showing no significant differences regarding the respiratory compensation point 69 . These findings highlight the relationship between systemic (i.e., ventilatory) and peripheral (i.e., oxygenation of locomotor and non-locomotor muscles) physiological breakpoints. In this sense, future studies should consider respiratory strategies associated with the NIRS technique to improve knowledge about the intensity of the training zone. Moreover, the IMW can attenuate muscle deoxygenation during exercise 35 and the NIRS technique can contribute to the monitoring of oxygenation in clinical practice 70 , especially in patients with exercise-induced ischemic pain caused by reduced blood flow to the lower extremities 71 .

Limitations and strengths
Despite the use of technologies with high-frequency signal acquisition, some limitations regarding our results must be addressed. First, the all-out run performed on a NMT has shown reliable results in the scientific literature 5,7-10,66,72 . However, we did not test the reproducibility of the four IMW interventions -although it is safe to say that they exhibited similar results in the 30 s all-out run tests (Fig. 2) with no differences in power running among the IMW loads second by second. We chose to investigate a classic anaerobic test (30 s all-out run), considering the aerobic component around ~ 18-20% in these efforts 7,13 . We observed an effect of IMW on the mechanical parameters, which did not result in greater muscle oxygenation differentiation. It is possible that by applying another slightly longer exercise protocol or repeated sprints the impact of IMW can be observed on both mechanical parameters (second by second) and physiological responses. Additionally, we did not use the gas analyzer to investigate the oxygen uptake due to our experimental design, nor investigated oxygenation of the inspiratory muscles. Still, we are aware that the association of NIRS measurements and VO 2 exchange would improve our data interpretation, but we have not tested whether this equipment can interfere with breathing pattern or breath frequency altering the isolated effects of IMW. Another limitation refers to our participants' characteristics, as only healthy active males, non-athletes performed the test. In future studies, we suggest the inclusion of female participants, the comparison of IMW with a shorter protocol and the analysis of effects in both high-performance athletes and non-athletes in different types of exercises, such as repeated-sprint effort.
The strengths of this study include: (i) the investigation of the inspiratory muscle strategy through a shorter protocol applied with methodological rigor, performed with practical and high-quality inspiratory devices, (ii) the running effort performed on a non-motorized treadmill able to identify, with high signal capture, minimal changes in mechanical variables during high-intensity exercise; (iii) monitoring oxygenation responses in different muscle groups, associated or not with respiratory strategies, allows a more integrative interpretation of this variable during effort and also during recovery.

Conclusion
In summary, different IMW loads with a shorter protocol (2 sets of 15 repetitions with a 1-min rest interval between sets and 2 min before exercise) applied on high-intensity running exercise suggested an improvement in performance corroborated by increased peak, mean and minimum mechanical values, but not in power and oxygenation assessed second by second. With respect to muscle oxygenation, these measurements demonstrated that the mechanisms by which IMW could possibly exert an effect on performance were not affected by these protocols, as all interventions showed similar and rapid adjustments of oxygenation responses during exercise demands. Interestingly, during passive recovery WU40 presented a pronounced TSI value for BB, indicating a greater availability of O 2 for lactate clearance in a tissue-dependent manner.

Data availability
The data that support the findings of this study are available from the corresponding author on reasonable request. Correspondence and requests for materials should be addressed to F.B.M.G.